Human map kinase homolog

ABSTRACT

The present invention provides nucleic acid and amino acid sequences that identify and encode a novel human map kinase homolog (SMAP) expressed in cells of the human stomach. The present invention also provides for PCR oligomers or hybridization probes for the detection of nucleotide sequences encoding SMAP or SMAP-like molecules, antisense molecules to the nucleotide sequences which encode SMAP, diagnostic tests based on SMAP encoding nucleic acid molecules, genetically engineered expression vectors and host cells for the production of purified SMAP, antibodies capable of binding specifically to SMAP, and agonists and inhibitors with specific binding activity for the polypeptide SMAP.

TECHNICAL FIELD

The present invention is in the field of molecular biology; moreparticularly, the present invention describes a nucleic acid sequenceand an amino acid sequence for a novel human MAP kinase homolog.

BACKGROUND ART

Mitogen-Activated Protein (MAP) Kinases

Mitogen-activated protein (MAP) kinases are a family of enzymes whichregulate intracellular signaling pathways. MAP kinases are importantmediators of signal transduction from cell surfaces to nuclei viaphosphorylation cascades. Several subgroups of MAP kinases have beendefined and each manifests different substrate specificities andresponds to various distinct extracellular stimuli. Thus, the MAP kinasesignaling pathways represent common mechanisms for signal transductionby which different extracellular stimuli generate distinct physiologicalresponses inside cells (Egan SE and Weinberg RA (1993) Nature365:781-783).

Various MAP kinase signaling pathways have been defined in mammaliancells as well as in yeast. In mammalian cells, the extracellular stimuliactivating the MAP kinase signaling pathways include epidermal growthfactor (EGF), ultraviolet light, hyperosmolar medium, heat shock,endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines suchas tumor necrosis factor (TNF) and interleukin-1 (IL-1). In the yeast,Saccharomyces cerevisiae, various MAP kinase signaling pathways areactivated by exposure to mating pheromone or hyperosmolar environmentsand during cell-wall construction, sporulation and mitosis.

There are at least three subgroups of MAP kinases in mammalian cells(Derijard B et al (1995) Science 267:682-5), and each subgroup isdistinguished by a tripeptide sequence motif. They are extracellularsignal-regulated protein kinase (ERK) characterized by Thr-Glu-Tyr,c-Jun amino-terminal kinase (JNK) characterized by Thr-Pro-Tyr, and p38kinase characterized by Thr-Gly-Tyr. The subgroups are activated by thedual phosphorylation of the threonine and tyrosine by MAP kinase kinaseslocated upstream of the phosphorylation cascade. Activated MAP kinasesphosphorylate other effectors downstream ultimately leading to changesinside the cell.

MAP Kinase Subgroup ERK

The ERK signal transduction pathway is activated via tyrosine kinasereceptors on the plasma membrane of the cell. When EGF or other growthfactors bind to the tyrosine receptors, they, in turn, bind tononcatalytic, src homology (SH) adaptor proteins (SH2-SH3-SH2) and aguanine nucleotide releasing protein. The latter reduces GTP andactivates Ras proteins, members of the large family of guaninenucleotide binding proteins (G-proteins). The activated Ras proteinsbind to a protein kinase C-Raf-1 and activate the Raf-1 proteins. Theactivated Raf-1 kinase subsequently phosphorylates MAP kinase kinaseswhich, in turn, activate MAP kinase ERKs by phosphorylating thethreonine and tyrosine residues of the ERKs.

ERKs are proline-directed protein kinases which phosphorylateSer/Thr-Pro motifs. In fact, cytoplasmic phospholipase A2 (cPLA2) andtranscription factor Elk-1 are substrates of the ERKs. The ERKsphosphorylate Ser₅₀₅ cPLA2 and cause an increase in its enzymaticactivity resulting in an increased release of arachidonic acid and theformation of lysophospholipids from membrane phospholipids. Likewise,phosphorylation of the transcription factor Elk-1 by ELK ultimatelyresults in increased transcriptional activity.

MAP Kinase Subgroup JNK

An analysis of a deduced primary sequence of the two isoforms of JNK, 46kDa and 55 kDa, reveals that they are distantly related to the ELKsubgroup. They are similarly activated by dual phosphorylation of Thrand Tyr, and the MKK4, MAP kinase kinases (Davis R (1994) TIBS19:470-473). The JNK signal transduction pathway can also be initiatedby ultraviolet light, osmotic stress, and the pro-inflammatorycytokines, TNF and IL-1. The Ras proteins may partially activate the JNKsignal transduction pathway. JNKs phosphorylate Ser₆₃ and Ser₇₃ in theamino-terminal domain of the transcription factor c-Jun which results inincreased transcriptional activity.

MAP Kinase Subgroup p38

An analysis of the cDNA sequence encoding p38 shows that p38 is a 41 kDprotein containing 360 amino acids. Its dual phosphorylation isactivated by the MAP kinase kinases, MKK3 and MKK4. The p38 signaltransduction pathway is also activated by heat shock, hyperosmolarmedium, IL-1or LPS endotoxin (Han J et al (1994) Science 265:808-811)produced by invading gram-negative bacteria. The human body reacts tothe invading bacteria by activating cells in the immune and inflammatorysystems to initiating the systemic response called sepsis. Sepsis ischaracterized by fever, chills, tachypnea, and tachycardia, and severecases may result in septic shock which includes hypotension and multipleorgan failure.

LPS may be thought of as a stress signal to the cell because it altersnormal cellular processes by inducing the release Of mediators such asTNF which has systemic effects. CD14 is aglycosylphosphatidyl-inositol-anchored membrane glycoprotein whichserves as an LPS receptor on the plasma membrane of cells of monocyticorigin. The binding of LPS to CD14 causes rapid protein tyrosinephosphorylation of the 44- and 42- or 40-kD isoforms of MAP kinases.Although they bind LPS, these MAP kinase isoforms do not appear tobelong to the p38 subgroup.

Other MAP Kinase Homologs

Recent research (Lee JC et al (1994) Nature 372:739-745) has revealedthat a new series of pyridinyl-imidazole compounds, which inhibitLPS-mediated human monocyte IL-1 and TNF-α production actually workthrough a pair of closely related MAP kinase homologs, termed cytokinesuppressive binding proteins (CSBPs). These compounds arecytokine-suppressive anti-inflammatory drugs (CSAIDs) which preventphosphorylation and subsequent cytokine biosynthesis. A comparison offragments of CSBP sequences with those of MAP kinases shows that genesencoding CSBPs are novel although related to protein serine/threoninekinases. It appears that CSBP proteins may be critical for cytokineproduction during human immune or inflammatory reactions.

Understanding the mechanism for blocking the specific kinase activitiesmay provide a new way of treating inflammatory illnesses. Likewise, athorough understanding of the various MAP kinase signaling pathways canenable scientists to better understand cell signaling in otherdevelopmental and disease processes. Identification of novel MAP kinasesprovides the opportunity to diagnose or intervene in such diseaseprocesses.

DISCLOSURE OF THE INVENTION

The subject invention provides a unique nucleotide sequence, hereindesignated in lower case, smap (SEQ ID NO:1) which encodes a novel humanMAP kinase protein, designated in upper case, SMAP (SEQ ID NO:2). ThecDNA encoding SMAP was identified and cloned using Incyte Clone No.214915 from a stomach cDNA library.

The invention also relates to the use of the nucleotide and amino acidsequences of SMAP, or its variants, in the diagnosis and treatment ofactivated or inflamed cells and/or tissues associated with itsexpression. Aspects of the invention include the antisense DNA of smap;cloning or expression vectors containing smap; host cells transformedwith the expression vector; a method for the production and recovery ofpurified SMAP from host cells; and purified protein, SMAP, which can beused to produce antibodies or identify inhibitors of the protein.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A and 1B display the alignment of the nucleotide sequence (SEQ IDNO:1) and amino acid sequence (SEQ ID NO:2) for human MAP kinase homologproduced using MacDNAsis software (Hitachi Software Engineering Co Ltd).

FIG. 2 shows the amino acid alignment between SMAP and mouse kinase,GenBank 531125 (locus MMU10871; Han et al. (1994) Science 265:808-810).

FIG. 3 shows the amino acid alignment between SMAP and the closelyrelated mitogen activated protein kinase homolog, GenBank 603917 (locusHUMCSBP1; Lee et al (1994) Nature 372:739-746). Alignments for FIGS. 2and 3 were produced using the INHERIT™ 670 Sequence Analysis System(Applied Biosystems, Foster City Calif).

MODES FOR CARRYING OUT THE INVENTION Definitions

As used herein, the lowercase letters, "smap", refer to a gene, cDNA ornucleic acid sequence for the novel human MAP kinase homolog while theuppercase letters, "SMAP", refer to the protein sequence encoded byhuman MAP kinase homolog.

The present invention provides a unique nucleotide sequence identifyinga novel MAP kinase homolog from human stomach cell, SEQ ID NO:1. Thecoding region of SEQ ID NO:1 begins at nucleotide 58 and ends atnucleotide 1156. Since SMAP is specifically involved with protectivecell signaling processes, the nucleic acid, protein, and antibodies areuseful in the study, diagnosis and treatment of conditions which affectthe stomach such as gastritis, ulcers, viral and bacterial infections,neoplasms and the like.

An "oligonucleotide" is a stretch of nucleotide residues which has asufficient number of bases to be used as an oligomer, amplimer or probein a polymerase chain reaction (PCR). Oligonucleotides are prepared fromgenomic or cDNA sequence and are used to amplify, confirm, or reveal thepresence of smap DNA or RNA in a particular cell or tissue.Oligonucleotides or oligomers comprise portions of a DNA sequence havingat least about 10 nucleotides and as many as about 50 nucleotides,preferably about 15 to 30 nucleotides.

"Probes" are nucleic acid sequences of variable length, preferablybetween 10 and 6,000 nucleotides, which may be chemically synthesized,naturally occurring, or recombinant single- or double-stranded nucleicacids. They are useful in the qualitative or quantitative detection ofthe same, a similar, or a complementary nucleic acid sequence.

"Reporter" molecules are chemical moieties used for labelling a nucleicor amino acid sequence. They include, but are not limited to,radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents. Reporter molecules associate with, establish the presence of,and may allow quantification of a particular nucleic or amino acidsequence.

A "portion" or "fragment" of a polynucleotide or nucleic acid comprisesall or any part of the nucleotide sequence having fewer nucleotides thanabout 6 kb, preferably fewer than about 1 kb which can be used as aprobe. Such probes may be labeled with reporter molecules using nicktranslation, Klenow fill-in reaction, PCR or other methods well known inthe art. After pretesting to optimize reaction conditions and toeliminate false positives, nucleic acid probes may be used in Southern,northern or in situ hybridizations to determine whether DNA or RNAencoding the protein is present in a biological sample, cell type,tissue, organ or organism.

"Recombinant nucleotide variants" are polynucleotides which encode SMAP.They may be synthesized by making use of the "redundancy" in the geneticcode. Various codon substitutions, such as the silent changes: whichproduce specific restriction sites or codon usage-specific mutations,may be introduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic host system,respectively.

"Linkers" are synthesized palindromic oligomers which create internalrestriction endonuclease sites.

"Chimeric" genes are polynucleotides which may be constructed byintroducing all or part of the nucleotide sequence of this inventioninto a vector containing additional nucleic acid sequence(s). Suchsequences may be expected to change any one (or more than one) of thefollowing SMAP characteristics: cellular location, distribution,ligand-binding affinities, interchain affinities, degradation/turnoverrate, signalling, etc.

"Active" refers to those forms, fragments, or domains of any SMAPpolypeptide which display the biologic and/or immunogenic activities ofany naturally occurring SMAP.

"Naturally occurring SMAP" refers to a polypeptide produced by cellswhich have not been genetically engineered and specifically contemplatesvarious polypeptides which arise from post-translational modifications.Such modifications of the polypeptide include but are not limited toacetylation, carboxylation, glycosylation, phosphorylation, lipidationand acylation.

"Derivative" refers to those polypeptides which have been chemicallymodified by such techniques as ubiquitination, labelling. (See above),pegylation (derivatization with polyethylene glycol), and chemicalinsertion or substitution of amino acids such as ornithine which do notnormally occur in human proteins.

"Recombinant polypeptide variant" refers to any polypeptide whichdiffers from naturally occurring SMAP by amino acid insertions,deletions and/or substitutions, created using recombinant DNAtechniques. Guidance in determining which amino acid residues may bereplaced, added or deleted without abolishing activities of interest maybe found by comparing the sequence of SMAP with that of relatedpolypeptides and minimizing the number of amino acid sequence changesmade in highly conserved regions.

Amino acid "substitutions" are defined as one for one amino acidreplacements. They are conservative in nature when the substituted aminoacid has similar structural and/or chemical properties. Examples ofconservative replacements are substitution of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine.

Amino acid "insertions" or "deletions" are changes to or within an aminoacid sequence. They typically fall in the range of about 1 to 5 aminoacids. The variation allowed in a particular amino acid sequence may beexperimentally determined by producing the peptide synthetically or bysystematically making insertions, deletions, or substitutions ofnucleotides in the smap sequence using recombinant DNA techniques.

A "signal or leader sequence" is a short amino acid sequence which canbe used, when desired, to direct the polypeptide through a membrane of acell. Such a sequence may be naturally present on the polypeptides ofthe present invention or provided from heterologous sources byrecombinant DNA techniques.

An "oligopeptide" is a short stretch of amino acid residues and may beexpressed from an oligonucleotide. It may be functionally equivalent toand the same length as (or considerably shorter than) a "fragment,""portion," or "segment" of a polypeptide. Such sequences comprise astretch of amino acid residues of at least about 5 amino acids and oftenabout 17 or more amino acids, typically at least about 9 to 13 aminoacids, and of sufficient length to display biologic and/or immunogenicactivity.

An "inhibitor" is a substance which retards or prevents a chemical orphysiological reaction or response. Common inhibitors include but arenot limited to antisense molecules, antibodies, and antagonists.

A "standard" is a quantitative or qualitative measurement forcomparison. It is based on a statistically appropriate number of normalsamples and is created to use as a basis of comparison when performingdiagnostic assays, running clinical trials, or following patienttreatment profiles.

"Animal" as used herein may be defined to include human, domestic (cats,dogs, etc.), agricultural (cows, horses, sheep etc) or test species(mouse, rat, rabbit, etc.)

Kinase nucleotide sequences have numerous applications in techniqueknown to those skilled in the art of molecular biology. These techniquesinclude the use of kinase sequences as hybridization probes, forchromosome and gene mapping, in the design of oligomers for PCR, and inthe production of sense or antisense nucleic acids, their chemicalanalogs and the like. These examples are well known and are not intendedto be limiting. Furthermore, the nucleotide sequences disclosed hereinmay be used in molecular biology techniques that have not yet beendeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known such as the triplet genetic code,specific base pair interactions, etc.

As a result of the degeneracy of the genetic code, a multitude ofkinase-encoding nucleotide sequences may be produced and some of thesewill bear only minimal homology to the endogenous sequence of any knownand naturally occurring kinase. This invention has specificallycontemplated each and every possible variation of nucleotide sequencethat could be made by selecting combinations based on possible codonchoices. These combinations are made in accordance with the standardtriplet genetic code as applied to the nucleotide sequence of naturallyoccurring kinases, and all such variations are to be considered as beingspecifically disclosed.

Although the nucleotide sequences which encode a specific kinase and itsderivatives or variants are preferably capable of identifying thenucleotide sequence of the naturally occurring kinase under optimizedconditions, it may be advantageous to produce smap possessing asubstantially different codon usage. Codons can be selected to increasethe rate of peptide expression in a particular prokaryotic or eukaryoticexpression host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding the kinase without alteringthe encoded amino acid sequence include the production of RNAtranscripts having more desirable properties, such as a longerhalf-life, than transcripts produced from the naturally occurringsequence.

Nucleotide sequences encoding a kinase may be joined to a variety ofother nucleotide sequences by means of well established recombinant DNAtechniques (Sambrook J et al (1989) Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.; orAusubel F M et al (1989) Current Protocols in Molecular Biology, JohnWiley & Sons, New York City). Useful nucleotide sequences for joining tothe kinase include an assortment of cloning vectors such as plasmids,cosmids, lambda phage derivatives, phagemids, and the like. Vectors ofinterest include vectors for replication, expression, probe generation,sequencing, and the like. In general, vectors of interest may contain anorigin of replication functional in at least one organism, convenientrestriction endonuclease sensitive sites, and selectable markers for oneor more host cell systems.

Another aspect of the subject invention provides for kinasehybridization probes which are capable of hybridizing with naturallyoccurring nucleotide sequences encoding kinases. The stringency of thehybridization conditions will determine whether the probe identifiesonly nuceotide sequence of that specific kinase or sequences of closelyrelated molecules. If such probes are used for the detection of relatedkinase encoding sequences, they should preferably contain at least 50%of the nucleotides from any of the sequence presented here.Hybridization probes of the subject invention may be derived from thenucleotide sequences of the SEQ ID NO:1 or from an isolated genomicsequence including untranslated regions such as promoters, enhancers andintrons. Such hybridization probes may be labeled with reportermolecules.

PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188provides additional uses for oligonucleotides based upon the kinasenucleotide sequence. Such oligomers may be of recombinant origin,chemically synthesized, or a mixture of both. Oligomers may comprise twonucleotide sequences employed under optimized conditions for tissuespecific identification or diagnostic use. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for identification of closelyrelated DNA or RNA sequences.

Full length genes may be cloned from known sequence using a new methoddisclosed in patent application Ser. No. 08/487,112 filed Jun. 7, 1995and hereby incorporated by reference, which employs XL-PCR(Perkin-Elmer, Foster City, Calif.) to amplify long pieces of DNA. Thismethod was developed to allow a Single researcher to process multiplegenes (up to 20 or more) at a time and to obtain an extended (possiblyfull-length) sequence within 6-10 days. It replaces current methodswhich use labeled probes to screen libraries and allow one researcher toprocess only about 3-5 genes in 14-40 days.

In the first step, which can be performed in about two days, primers aredesigned and synthesized based on a known partial sequence. In step 2,which takes about six to eight hours, the sequence is extended by PCRamplification of a selected library. Steps 3 and 4, which take about oneday, are purification of the amplified cDNA and its ligation into anappropriate vector, respectively. Step 5, which takes about one day,involves transforming and growing up host bacteria. In step 6, whichtakes approximately five hours, PCR is used to screen bacterial clonesfor extended sequence. The final steps, which take about one day,involve the preparation and sequencing of selected clones. If the fulllength cDNA has not been obtained, the entire procedure is repeatedusing either the original library or some other preferred library. Thepreferred library may be one that has been size-selected to include onlylarger cDNAs or may consist of single or combined commercially availablelibraries, eg. lung, liver, heart and brain from Gibco/BRL (GaithersburgMd.). The cDNA library may have been prepared with oligo d(T) or randomprimers. The advantage of using random primed libraries is that theywill have more sequences which contain 5' ends of genes. A randomlyprimed library may be particularly useful if an oligo d(T) library doesnot yield a complete gene. Obviously, the larger the protein, the lesslikely it is that the complete gene will be found in a single plasmid.

Other means of producing specific hybridization probes for kinasesinclude the cloning of the cDNA sequences into vectors for theproduction of mRNA probes. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7 or SP6 andlabeled nucleotides.

It is possible to produce a DNA sequence, or portions thereof, entirelyby synthetic chemistry. After synthesis, the nucleic acid sequence canbe inserted into any of the many available DNA vectors and theirrespective host cells using techniques which are well known in the art.Moreover, synthetic chemistry may be used to introduce mutations intothe nucleotide sequence. Alternately, a portion of sequence in which amutation is desired can be synthesized and recombined with a portion ofan existing genomic or recombinant sequence.

The kinase nucleotide sequences can be used individually, or in panels,in an assay to detect inflammation or disease associated with abnormallevels of kinase expression. The nucleotide sequence is added to afluid, cell or tissue sample from a patient under hybridizingconditions. After an incubation period, the sample is washed with acompatible fluid which optionally contains a reporter molecule whichwill bind the specific nucleotide. After the compatible fluid is rinsedoff, the reporter molecule is quantitated and compared with a standardfor that fluid, cell or tissue. If kinase expression is significantlydifferent from the standard, the assay indicates the presence ofinflammation or disease.

This same assay, combining a sample with the nucleotide sequence, isapplicable in evaluating the efficacy of a particular therapeutictreatment regime. It may be used in animal studies, in clinical trials,or in monitoring the treatment of an individual patient. First, standardexpression must be established for use as a basis of comparison. Second,samples from the animals or patients affected by the disease arecombined with the nucleotide sequence to evaluate the deviation from thestandard or normal profile. Third, an existing therapeutic agent isadministered, and a treatment profile is generated. The assay isevaluated to determine whether the profile progresses toward or returnsto the standard pattern. Successive treatment profiles may be used toshow the effects of treatment over a period of several days or overseveral months.

The cDNA for human MAP kinase can also be used to design hybridizationprobes for mapping the native genomic sequence. The sequence may bemapped to a particular chromosome or to a specific region of thechromosome using well known techniques. These include in situhybridization to chromosomal spreads (Verma et al (1988) HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New YorkCity), flow-sorted chromosomal preparations, or artificial chromosomeconstructions such as yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions or singlechromosome cDNA libraries.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers are invaluable in extending genetic maps. Examples of geneticmap data can be found in the 1994 Genome Issue of Science (265:1981f).Often the placement of a gene on the chromosome of another mammalianspecies may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New nucleotide sequences canbe assigned to chromosomal subregions by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once a disease orsyndrome, such as ataxia telangiectasia (AT), has been crudely localizedby genetic linkage to a particular genomic region, for example, AT to11q22-23 (Gatti et al (1988) Nature 336:577-580), any sequences mappingto that area may represent genes for further investigation of AT. Thenucleotide sequence of the subject invention may also be used to detectdifferences in gene sequence between normal and carrier or affectedindividuals.

Nucleotide sequences encoding a particular kinase may be used to producepurified oligopeptide using well known methods of recombinant DNAtechnology. Goeddel (1990, Gene Expression Technology, Methods andEnzymology, Vol 185, Academic Press, San Diego Calif.) is one among manypublications which teach expression of an isolated nucleotide sequence.The oligopeptide may be expressed in a variety of host cells, eitherprokaryotic or eukaryotic. Host cells may be from the same species fromwhich the nucleotide sequence was derived or from a different species.Advantages of producing an oligonucleotide by recombinant DNA technologyinclude obtaining adequate amounts of the protein for purification andthe availability of simplified purification procedures.

Cells transformed with a kinase nucleotide sequence may be culturedunder conditions suitable for the expression and recovery of theoligopeptide from cell culture. The oligopeptide produced by arecombinant cell may be secreted or may be contained intracellularlydepending on the sequence and the genetic construction used. In general,it is more convenient to prepare recombinant proteins in secreted form.Purification steps vary with the production process and the particularprotein produced. Often an oligopeptide can be produced from a chimericnucleotide sequence. This is accomplished by ligating the kinasesequence to a nucleic acid sequence encoding a polypeptide domain whichwill facilitate protein purification (Kroll D J et al (1993) DNA CellBiol 12:441-53).

In addition to recombinant or chimeric production, kinase fragments maybe produced by direct peptide synthesis using solid-phase techniques(Stewart et al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, SanFrancisco Calif.; Merrifield J (1963) J Am Chem Soc 85:2149-2154).Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer in accordance with the instructionsprovided by the manufacturer. Additionally a particular kinase sequence,or any part thereof, may be mutated during chemical synthesis, combinedusing chemical methods with other kinase sequence(s), and used in anappropriate vector and host cell to produce a polypeptide.

Although the amino acid sequence or oligopeptide used for antibodyinduction does not require biological activity, it must be antigenic andconsist of at least five amino acids and preferably at least 10 aminoacids. Short stretches of amino acid sequence may be fused with those ofanother protein such as keyhole limpet hemocyanin, and the chimericpeptide used for antibody production.

Antibodies specific for SMAP may be produced by inoculation of anappropriate animal with an antigenic fragment of the peptide. Anantibody is specific for SMAP if it is produced against an epitope ofthe polypeptide and binds to at least part of the natural or recombinantprotein. Antibody production includes not only the stimulation of animmune response by injection into animals, but also analogous processessuch as the production of synthetic antibodies, the screening ofrecombinant immunoglobulin libraries for specific-binding molecules(Orlandi R et al (1989) PNAS 86:3833-3837, or Huse W D et al (1989)Science 256:1275-1281), or the in vitro stimulation of lymphocytepopulations. Current technology (Winter G and Milstein C (1991) Nature349:293-299) provides for a number of highly specific binding reagentsbased on the principles of antibody formation. These techniques may beadapted to produce molecules which specifically bind SMAPs.

The examples below are provided to illustrate the subject invention.These examples are provid by way of illustration and are not includedfor the purpose of limiting the invention.

INDUSTRIAL APPLICABILITY

I Isolation of mRNA and Construction of the cDNA Library

The partial cDNA sequence for the human MAP kinase homolog was initiallyidentified in Incyte Clone 214915 among the sequences comprising thehuman stomach cell library, patent application Ser. No. 08/385,268,filed 7 Feb. 1995, disclosed herein by reference. The normal stomachtissue used for this library was obtained from the Keystone Skin Bank,International Institute for the Advancement of Medicine (Exton Pa.).

Five grams of normal stomach tissue from a 55 year old male (KSP93-B72)was flash frozen, ground in a mortar and pestle, and lysed immediatelyin buffer containing guanidinium isothiocyanate. Lysis was followed bycentrifugation through cesium chloride, incubation with DNase andethanol precipitation.

The RNA was sent to Stratagene (La Jolla Calif.) and oligo d(T) primingwas used to prepare the cDNA library. Synthetic linkers were ligatedonto the cDNA molecules, and they were inserted into the Uni-ZAP™ vectorsystem (Stratagene).

II Isolation of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which the host bacterial strain wasco-infected with both the library phage and an f1 helper phage.Polypeptides or enzymes derived from both the library-containing phageand the helper phage nicked the DNA, initiated new DNA synthesis fromdefined sequences on the target DNA, and created a smaller, singlestranded circular phagemid DNA molecule that included all DNA sequencesof the pBluescript phagemid and the cDNA insert. The phagemid DNA wasreleased from the cells, purified, and used to reinfect fresh host cells(SOLR, Stratagene) where double-stranded phagemid DNA was produced.

Phagemid DNA was purified using the QIAWELL-8™ Plasmid PurificationSystem (QIAGEN Inc, Chatsworth Calif.). This product lyses bacterialcells and allows the isolation of highly purified phagemid DNA usingQIAGEN anion-exchange resin particles in a multiwell format. The DNA waseluted from the purification resin and prepared for DNA sequencing andother analytical manipulations.

An alternate method of purifying phagemid utilizes the Miniprep Kit(Catalog No. 77468; Advanced Genetic Technologies Corp, GaithersburgMd.). The kit has a 96-well format and provides enough reagents for 960purifications. The recommended protocol is employed except for thefollowing changes. First, each of the 96 wells is filled with 1 ml ofsterile terrific broth (LIFE TECHNOLOGIES™, Gaithersburg Md.) containingcarbenicillin at 25 mg/L and glycerol at 0.4%. The bacteria areintroduced into the wells, cultured for 24 hours and lysed with 60 μl oflysis buffer. The block is centrifuged at 2900 rpm for 5 minutes andthen the contents of the block are added to the primary filter plate. Anoptional step of adding isopropanol to the TRIS buffer is not routinelyperformed. Following the last step in the protocol, samples aretransferred to a Beckman 96-well block for storage.

III Sequencing of cDNA Clones

The cDNA inserts from random isolates of the stomach library weresequenced in part. Methods for DNA sequencing are well known in the artand employ such enzymes as SEQUENASE® (US Biochemical Corp, Cleveland,Ohio) or Taq polymerase. Methods to extend the DNA from anoligonucleotide primer annealed to the DNA template of interest havebeen developed for the use of both single- and double-strandedtemplates. The chain termination reaction product were separated usingelectrophoresis and urea-acrylamide gels and detected either byautoradiography with radionuclide-labeled precursors or by fluorescentor chromogenic labelling. Recent improvements in mechanized reactionpreparation, sequencing and analysis using the latter methods havepermitted expansion in the number of sequences determined per day. Themachines used in these processes include the Catalyst 800, HamiltonMicro Lab 2200 (Hamilton, Reno Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown Mass.) and the Applied Biosystems 377 and 373 DNAsequencers.

IV Homology Searching of cDNA Clones and Deduced Proteins

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems and incorporated intothe INHERIT™ 670 Sequence Analysis System. In this algorithm, PatternSpecification Language (developed by TRW Inc, Los Angeles Calif.) wasused to determine regions of homology. The three parameters thatdetermine how the sequence comparisons run were window size, windowoffset, and error tolerance. Using a combination of these threeparameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions, of homology from chance matches. Smith-Waterman alignments wereused to display the results of the hornology search.

Peptide and protein sequence hornologies were ascertained using theINHERIT™ 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

Alternatively, BLAST, which stands for Basic Local Alignment SearchTool, is used to search for local sequence: alignments (Altschul SF(1993) J Mol Evol 36:290-300; Altschul, SF et al (1990) J Mol Biol215:403-10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. Whereas it is ideal for matcheswhich do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP).

An HSP consists of two sequence, fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

V Extension of the cDNA to Full Length

Analysis of the INHERIT™ result from the randomly picked and sequencedportions of clones from the stomach library identified Incyte 214915 asa homolog of MAP kinase. The cDNA of Incyte 214915 was extended to fulllength using a modified XL-PCR (Perkin Elmer) procedure. Primers weredesigned based on the known sequence; one primer was synthesized toinitiate extension in the antisense direction (XLR) and the other toextend sequence in the sense direction (XLF). The primers allowed thesequence to be extended "outward" generating amplicons containing new,unknown nucleotide sequence for the gene of interest. The primers weredesigned using Oligo 4.0 (National Biosciences Inc, Plymouth Minn.) tobe 22-30 nucleotides in length, to have a GC content Of 50% or more, andto anneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations was avoided.

The stomach cDNA library was used as a template, and XLR=AAG ACA TCC AGGAGC CCA ATG AC and XLF=AGG TGA TCC TCA GCT GGA TGC AC primers were usedto extend and amplify the 214915 sequence. By following the,instructions for the XL-PCR kit and thoroughly mixing the enzyme andreaction mix, high fidelity amplification is obtained. Beginning with 25pMol of each primer and the recommended concentrations of all othercomponents of the kit, PCR was performed using the Peltier thermalcycler (MJ PTC200; MJ Research, Watertown Mass.) and the followingparameters:

Step 1 94° C. for 60 sec (initial denaturation)

Step 2 94° C. for 15 sec

Step 3 65° C. for 1 min

Step 4 68° C. for 7 min

Step 5 Repeat step 2-4 for 15 additional times

Step 6 94° C. for 15 sec

Step 7 65° C. for 1 min

step 8 68° C. for 7 min+15 sec/cycle

step 9 Repeat step 6-8 for 11 additional times

Step 10 72° C. for 8 min

Step 11 4° C. (and holding)

At the end of 28 cycles, 50 μl of the reaction mix was removed; and theremaining reaction mix was run for an additional 10 cycles as outlinedbelow:

Step 1 94° C. for 15 sec

Step 2 65° C. for 1 min

Step 3 68° C. for (10 min+15 sec)/cycle

Step 4 Repeat step 1-3 for 9 additional times

Step 5 72° C. for 10 min

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Although all extensions potentially contain a full length gene, some ofthe largest products or bands were selected and cut out of the gel.Further purification involved using a commercial gel extraction methodsuch as QIAQuick™ (QIAGEN Inc). After recovery of the DNA, Klenow enzymewas used to trim single-stranded, nucleotide overhangs creating bluntends which facilitated religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer. Then; 1 μT4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coil cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture, was plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing carbenicillin at 25 mg/L. The following day, 12 colonies wererandomly picked from each plate and cultured in 150 μl of liquidLB/carbenicillin medium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture was transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each samplewas transferred into a PCR array.

For PCR amplification, 15 μl of concentrated PCR reaction mix (1.33X)containing 0.75 units of Taq polymerase, a vector primer and one or bothof the gene specific primers used for the extension reaction were addedto each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec

Step 2 94° C. for 20 sec

step 3 55° C. for 30 sec

step 4 72° C. for 90 sec

Step 5 Repeat steps 2-4 for an additional 29 times

Step 6 72° C. for 180 sec

Step 7 4° C. (and holding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated-into plasmid and sequenced.

When the three possible amino acid translations of the full length cDNAsequence were searched against protein databases such as SwissProt andPIR, no exact matches were found. FIG. 1 shows the nucleotide and aminoacid sequences for human MAP kinase homolog. The alignment of the aminoacid sequence for SMAP (SEQ ID NO:2) with MMU10871 (GI 531125, SEQ IDNO:3) and HUMCSBP1(GI 603917) are shown in FIGS. 2 and 3, respectively.

VI Sense or Antisense Molecules

Knowledge of the correct cDNA sequence of any particular kinase, or partthereof, enables its use as a tool in sense or antisense technologiesfor the investigation of gene function. Oligonucleotides, from genomicor cDNAs, comprising either the sense or the antisense strand of thecDNA sequence is used in vitro or in vivo to inhibit expression. Suchtechnology is now well known in the art, and oligonucleotides or otherfragments are designed from various locations along the sequences. Thegene of interest is turned off in the short term by transfecting a cellor tissue with expression vectors which flood the cell with sense orantisense sequences until all copies of the vector are disabled byendogenous nucleases. Stable transfection of appropriate germ line cellsor a zygote with a vector containing the fragment produces a transgenicorganism (U.S. Pat. No. 4,736,866, 12 Apr. 1988), whose cells produceenough copies of the sense or antisense sequence to significantlycompromise or entirely eliminate normal activity of the particularkinase gene. Frequently, the function of the gene is ascertained byobserving behaviors such as lethality, loss of a physiological pathway,changes in morphology, etc. at the intracellular, cellular, tissue ororganismal level.

In addition to using fragments constructed to interrupt transcription ofthe open reading frame, modifications of gene expression, are obtainedby designing antisense sequences to promoters, enhancers, introns, oreven to transacting regulatory genes. Similarly, inhibition is achievedusing Hogeboom base-pairing methodology, also known as "triple helix"base pairing.

VII Expression of SMAP

Expression of smap is accomplished by subcloning the cDNAs intoappropriate expression vectors and transfecting the vectors into anappropriate expression hosts. In this particular case, the cloningvector previously used for the generation of the tissue library alsoprovide for direct expression of smap sequences in E. coli. Upstream ofthe cloning site, this vector contains a promoter for β-galactosidase,followed by sequence containing the amino-terminal Met and thesubsequent 7 residues of β-galactosidase. Immediately following theseeight residues is an engineered bacteriophage promoter useful forartificial priming and transcription and a number of unique restrictionsites, including Eco RI, for cloning.

Induction of the isolated, transfected bacterial strain with IPTG usingstandard methods produces a fusion protein corresponding to the firstseven residues of β-galactosidase, about 5 to 15 residues whichcorrespond to linker, and the peptide encoded within the cDNA. SincecDNA clone inserts are generated by an essentially random process, thereis one chance in three that the included cDNA lies in the correct framefor proper translation. If the cDNA is not in the proper reading frame,it is obtained by deletion or insertion of the appropriate number ofbases by well known methods including in vitro mutagenesis, digestionwith exonuclease III or mung bean nuclease, or oligonucleotide linkerinclusion.

The smap cDNA is shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonucleotide linkercontaining cloning sites as well as a segment of DNA sufficient tohybridize to stretches at both ends of the target cDNA (25 bases) issynthesized chemically by standard methods. These primers are then usedto amplify the desired gene segments by PCR. The resulting new genesegments are digested with appropriate restriction enzymes understandard conditions and isolated by gel electrophoresis. Alternately,similar gene segments are produced by digestion of the cDNA withappropriate restriction enzymes and filling in the missing gene segmentswith chemically synthesized oligonucleotides. Segments of the codingsequence from more than one gene are ligated together and cloned inappropriate vectors to optimize expression of recombinant sequence.

Suitable expression hosts for such chimeric molecules include but arenot limited to mammalian cells such as Chinese Hamster Ovary (CHO) andhuman 293 cells, insect cells such as Sf9 cells, yeast cells such asSaccharomyces cerevisiae, and bacteria such as E. coli. For each ofthese cell systems, a useful expression vector includes an origin ofreplication to allow propagation in bacteria and a selectable markersuch as the β-lactamase antibiotic resistance gene to allow selection inbacteria. In addition, the vectors include a second selectable markersuch as the neomycin phosphotransferase gene to allow selection intransfected eukaryotic host cells. Vectors for use in eukaryoticexpression hosts usually require RNA processing elements such as 3'polyadenylation sequences if such are not part of the cDNA of interest.

Additionally, the vector contains promoters or enhancers which increasegene expression. Such promoters are host specific and include MMTV,SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGHpromoters for yeast. Transcription enhancers, such as the rous sarcomavirus (RSV) enhancer, is used in mammalian host cells. Once homogeneouscultures of recombinant cells are obtained through standard culturemethods, large quantities of recombinantly produced SMAP are recoveredfrom the conditioned medium and analyzed using chromatographic methodsknown in the art.

VIII Isolation of Recombinant SMAP

SMAP is expressed as a chimeric protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification-facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase (Invitrogen) between the purification domain and thesmap sequence is useful to facilitate purification of SMAP.

IX Production of SMAP Specific Antibodies

Two approaches are utilized raise antibodies to SMAP, and each approachis useful for generating either polyclonal or monoclonal antibodies. Inone approach, denatured protein from the reverse phase HPLC separationis obtained in quantities up to 75 mg. This denatured protein is used toimmunize mice or rabbits using standard protocols; about 100 microgramsare adequate for immunization of a mouse, while up to 1 mg might be usedto immunize a rabbit. For identifying mouse hybridomas, the denaturedprotein is radioiodinated and used to screen potential murine B-cellhybridomas for those which produce antibody. This procedure requiresonly small quantities of protein, such that 20 mg would be sufficientfor labeling and screening of several thousand clones.

In the second approach, the amino acid sequence of SMAP, as deduced fromtranslation of the cDNA, is analyzed to determine regions of highimmunogenicity. Oligopeptides comprising appropriate hydrophilic regionsare synthesized and used in suitable immunization protocols to raiseantibodies. Analysis to select appropriate epitopes is described byAusubel F M et al (supra). The optimal amino acid sequences forimmunization are usually at the C-terminus, the N-terminus and thoseintervening, hydrophilic region of the polypeptide which are likely tobe exposed to the external environment when the protein is in itsnatural conformation.

Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH,Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS; Ausubel F M et al, supra). If necessary, a cysteine is introducedat the N-terminus of the peptide to permit coupling to KLH. Rabbits areimmunized with the peptide-KLH complex in complete Freund's adjuvant.The resulting antisera are tested for antipeptide activity by bindingthe peptide to plastic, blocking with 1% bovine serum albumin, reactingwith antisera, washing and reacting with labeled (radioactive orfluorescent), affinity purified, specific goat anti-rabbit IgG.

Hybridomas are prepared and screened using standard techniques.Hybridomas of interest are detected by screening with labeled: SMAP toidentify those fusions producing the monoclonal antibody with thedesired specificity. In a typical protocol, wells of plates (FAST;Becton-Dickinson, Palo Alto, Calif.) are coated during incubation withaffinity purified, specific rabbit anti-mouse (or suitable anti-specieslg) antibodies at 10 mg/ml. The coated wells are blocked with 1% BSA,washed and incubated with supernatants from hybridomas. After washingthe wells are incubated with labeled SMAP at 1 mg/ml. Supernatants withspecific antibodies bind more labeled SMAP than is detectable in thebackground. Then clones producing specific antibodies are expanded andsubjected to two cycles of cloning at limiting dilution (1 cell/3wells). Cloned hybridomas are injected into pristane-treated mice toproduce ascites, and monoclonal antibody is purified from mouse asciticfluid by affinity chromatography on Protein A. Monoclonal antibodieswith affinities of at least 10⁸ /M, preferably 10⁹ to 10¹⁰ or stronger,are typically made by standard procedures as described in Harlow andLane (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; and in Goding (1986) MonoclonalAntibodies: Principles and Practice, Academic Press, New York City, bothincorporated herein by reference:

X Diagnostic Test Using SMAP Specific Antibodies

Particular SMAP antibodies are useful for investigation of various formsof stomach conditions characterized by differences in the amount ordistribution of SMAP. Given the usual role of MAP kinases, SMAP from thehuman stomach library appears to be upregulated in its characteristicinvolvement in immune protection or defense.

Diagnostic tests for SMAP include methods utilizing the antibody and alabel to detect SMAP in human body fluids, membranes, cells, tissues andantibodies of the present invention are used with or withoutmodification. Frequently, the polypeptides and antibodies are labeled byjoining them, either covalently or noncovalently, with a substance whichprovides for a detectable signal. A wide variety of labels andconjugation techniques are known and have been reported extensively inboth the scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentagents, chemiluminescent agents, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins are produced as shown inU.S. Pat. No. 4,816,567, incorporated herein by reference.

A variety of protocols for measuring soluble or membrane-bound SMAP,using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson SMAP is preferred, but a competitive binding assay may be employed.These assays are described, among other places, in Maddox, D E et al(1983, J Exp Med 158:1211).

XI Purification of Native SMAP Using Specific Antibodies

Native or recombinant SMAP is purified by immunoaffinity chromatographyusing antibodies specific for SMAP. In general, an immunoaffinity columnis constructed by covalently coupling the anti-SMAP antibody to anactivated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated Sepharose (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such immunoaffinity columns are utilized in the purification of SMAP bypreparing a fraction from cells containing SMAP in a soluble form. Thispreparation is derived by solubilization of whole cells or of asubcellular fraction obtained via differential centrifugation (with orwithout addition of detergent) or by other methods well known in theart. Alternatively, soluble SMAP containing a signal sequence issecreted in useful quantity into the medium in which the cells aregrown.

A soluble SMAP-containing preparation is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of SMAP (eg, high ionic strength buffers in thepresence of detergent). Then, the column is eluted under conditions thatdisrupt antibody/SMAP binding (eg, a buffer of pH 2-3 or a highconcentration of a chaotrope such as urea or thiocyanate ion), and SMAPis collected.

XII Drug Screening

This invention is particularly useful for screening therapeuticcompounds by using SMAP or binding fragments thereof in any of a varietyof drug screening techniques. The polypeptide or fragment employed insuch a test is either free in solution, affixed to a solid support,borne on a cell surface or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the polypeptide orfragment. Drugs are screened against such transformed cells incompetitive binding assays. Such cells, either in viable or fixed form,are used for standard binding assays. One measures, for example, theformation of complexes between SMAP and the agent being tested.Alternatively, one can examine the diminution in complex formationbetween SMAP and a receptor caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which affect signal transduction. These methodscomprise contacting such an agent with SMAP polypeptide or a fragmentthereof and assaying (i) for the presence of a complex between the agentand the SMAP polypeptide or fragment, or (ii) for the presence of acomplex between the SMAP polypeptide or fragment and the, cell, bymethods well known in the art. In such competitive binding assays, theSMAP polypeptide or fragment is typically labeled. After suitableincubation, free SMAP polypeptide or fragment is separated from thatpresent in bound form, and the amount of free or uncomplexed label is ameasure of the ability of the particular agent to bind to SMAP or tointerfere with the SMAP and agent complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the SMAP polypeptidesand is described in detail in European Patent Application 84/03564,published on Sept. 13, 1984, incorporated herein by reference. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with SMAP polypeptideand washed. Bound SMAP polypeptide is then detected by methods wellknown in the art. Purified SMAP may also be coated directly onto platesfor use in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding SMAPspecifically compete with a test compound for binding to SMAPpolypeptides or fragments thereof. In this manner, the antibodies areused to detect the presence of any peptide which shares one or moreantigenic determinants with SMAP.

XIII Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, e.g., agonists, antagonists, or inhibitors. Any ofthese examples are used to fashion drugs which are more active or stableforms of the polypeptide or which enhance or interfere with the functionof a polypeptide in vivo (Hodgson J (1991), Bio/Technology 9:19-21,incorporated herein by reference).

In one approach, the three-dimensional structure of a protein ofinterest, or of a protein-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide is gained by modeling based onthe structure of homologous proteins. In both cases, relevant structuralinformation is used to design efficient inhibitors. Useful examples ofrational drug design include molecules which have improved activity orstability as shown by Braxton S and Wells J A (1992 Biochemistry31:7796-7801) or which act as inhibitors, agonists, or antagonists ofnative peptides as shown by Athauda S B et al (1993 J Biochem113:742-746), incorporated herein by reference.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve it crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design is based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(ant-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids isexpected to be an analog of the original receptor. The anti-ids is thenused to identify and isolate peptides from banks of chemically orbiologically,produced peptides. The isolated peptides then act as thepharmacore.

By virtue of the present invention, sufficient amount of polypeptide ismade available to perform such analytical studies as X-raycrystallography. In addition, knowledge of the SMAP amino acid sequenceprovided herein provides guidance to those employing computer modelingtechniques in place of or in addition to x-ray crystallography.

XIV Identification of Other Members of the Signal Transduction Complex

The inventive purified SMAP is a research tool for identification,characterization and purification of interacting or signal transductionpathway proteins. Radioactive labels are incorporated into SMAP byvarious methods known in the art and used to capture either soluble ormembrane-bound molecules. A preferred method involves labeling theprimary amino groups in SMAP with ¹²⁵ l Bolton-Hunter reagent (Bolton, AE and Hunter, W M (1973) Biochem J 133: 529). This reagent has been usedto label various molecules without concomitant loss of biologicalactivity (Hebert C A et al (1991) J Biol Chem 266: 18989; McColl S et al(1993) J Immunol 150:4550-4555). Membrane-bound molecules are incubatedwith the labeled SMAP molecules, washed to removed unbound molecules,and the SMAP complex is quantified. Data obtained using differentconcentrations of SMAP are used to calculate values for the number,affinity, and association of SMAP complex.

Labeled SMAP is also useful as a reagent for the purification ofmolecules with which SMAP interacts. In one embodiment of affinitypurification, SMAP is covalently coupled to a chromatography column.Cells and their membranes are extracted, SMAP is removed and variousSMAP-free subcomponents are passed over the column. Molecules bind tothe column by virtue of their SMAP affinity. The SMAP-complex isrecovered from the column, dissociated and the recovered molecule issubjected to N-terminal protein sequencing. This amino acid sequence isthen used to identify the captured molecule or to design degenerateoligonucleotide probes for cloning its gene from an appropriate cDNAlibrary.

In another alternate method, antibodies are raised against SMAP,specifically monoclonal antibodies. The monoclonal antibodies arescreened to identify those which inhibit the binding of labeled SMAP.These monoclonal antibodies are then used in affinity purification orexpression cloning of associated molecules.

Other soluble binding molecules are identified in a similar manner.Labeled SMAP is incubated with extracts or other appropriate materialsderived from stomach or other gastrointestinal mucosa. After incubation,SMAP complexes (which are larger than the lone SMAP molecule) areidentified by a sizing technique such as size exclusion chromatographyor density gradient centrifugation and are purified by methods known inthe art. The soluble binding protein(s) are subjected to N-terminalsequencing to obtain information sufficient for database identification,if the soluble protein is known, or for cloning, if the soluble proteinis unknown.

XV Use and Administration of Antibodies, Inhibitors, Receptors orAntagonists of SMAP

Antibodies, inhibitors, receptors or antagonists of SMAP (or othertreatments to limit signal transduction, TST) provide different effectswhen administered therapeutically. TSTs are formulated in a nontoxic,inert, pharmaceutically acceptable aqueous carrier medium preferably ata pH of about 5 to 8, more preferably 6 to 8, although the pH may varyaccording to the characteristics of the antibody, inhibitor, orantagonist being formulated and the condition to be treated.Characteristics of TSTs include solubility of the molecule, half-lifeand antigenicity/immunogenicity; these and other characteristics aid indefining an effective carrier. Native human proteins are preferred asTSTs, but organic or synthetic molecules resulting from drug screens areequally effective in particular situations.

TSTs are delivered by known routes of administration including but notlimited to topical creams and gels; transmucosal spray and aerosol;transdermal patch and bandage; injectable, intravenous and lavageformulations; and orally administered liquids and pills particularlyformulated to resist stomach acid and enzymes. The particularformulation, exact dosage, and route of administration are determined bythe attending physician and vary according to each specific situation.

Such determinations are made by considering multiple variables such asthe condition to be treated, the TST to be administered, and thepharmacokinetic profile of the particular TST. Additional factors whichare taken into account include disease state (e.g. severity) of thepatient, age, weight, gender, diet, time and frequency ofadministration, drug combination, reaction sensitivities, andtolerance/response to therapy. Long acting TST formulations might beadministered every 3 to 4 days, every week, or once every two weeksdepending on half-life and clearance rate of the particular TST.

Normal dosage amounts vary from 0.1 to 100,000 micrograms, up to a totaldose of about 1 g, depending upon the route of administration. Guidanceas to particular dosages and methods of delivery is provided in theliterature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. Thoseskilled in the art employ different formulations for different TSTs.Administration to cells such as nerve cells necessitates delivery in amanner different from that to other cells such as vascular endothelialcells.

It is contemplated that conditions or diseases which trigger defensivesignal transduction may precipitate damage that is treatable with TSTs.These conditions or diseases are specifically diagnosed by the testsdiscussed above, and such testing should be performed in suspected casesof stomach conditions such as gastritis, ulcers, viral and bacterialinfections, or neoplasms associated with abnormal signal transduction.

All publications and patents mentioned the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention are apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1851 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Stomach                                                          (B) CLONE: 214915                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCCCGTTGGGCCGCGAACGCAGCCGCCACGCCGGGGCCGCCGAGATCGGGTGCCCGGGAT60                GAGCCTCATCCGGAAAAAGGGCTTCTACAAGCAGGACGTCAACAAGACCGCCTGGGAGCT120               GCCCAAGACCTACGTGTCCCCGACGCACGTCGGCAGCGGGGCCTATGGCTCCGTGTGCTC180               GGCCATCGACAAGCGGTCAGGGGAGAAGGTGGCCATCAAGAAGCTGAGCCGACCCTTTCA240               GTCCGAGATCTTCGCCAAGCGCGCCTACCGGGAGCTGCTGTTGCTGAAGCACATGCAGCA300               TGAGAACGTCATTGGGCTCCTGGATGTCTTCACCCCAGCCTCCTCCCTGGAACTTCTATG360               ACTTCTACCTGGTGATGCCCTTCATGCAGACGGATCTGCAGAAGATCATGGGGATGGAGT420               TCAGTGAGGAGAAGATCCAGTACCTGGTGTATCAGATGCTCAAAGGCCTTAAGTACATCC480               ACTCTGCTGGGGTCGTGCACAGGGACCTGAAGCCAGGCAACCTGGCTGTGAATGAGGACT540               GTGAACTGAAGATTCTGGATTTGGGGCTGGCGCGACATGCAGACGCCGAGATGACTGGCT600               ACGTGGTGACCCGCTGGTACCGAGCCCCCGAGGTGATCCTCAGCTGGATGCACTACAACC660               AGACAGTGGACATCTGGTCTGTGGGCTGTATCATGGCAGAGATGCTGACAGGGAAAACTC720               TGTTCAAGGGGAAAGATTACCTGGACCAGCTGACCCAGATCCTGAAAGTGACCGGGGTGC780               CTGGCACGGAGTTTGTGCAGAAGCTGAACGACAAAGCGGCCAAATCCTACATCCAGTCCC840               TGCCACAGACCCCCAGGAAGGATTTCACTCAGCTGTTCCCACGGGCCAGCCCCCAGCCTG900               CGGACCTGCTGGAGAAGATGCTGGAGCTAGACGTGGACAAGCGCCTGACGGCCGCGCAGG960               CCCTCACCCATCCCTTCTTTGAACCCTTCCGGGACCCTGAGGAAGAGACGGAGGCCCAGC1020              AGCCGTTTGATGATTCCTTAGAACACGAGAAACTCACAGTGGATGAATGGAAGCAGCACA1080              TCTACAAGGAGATTGTGAACTTCAGCCCCATTGCCCGGAAGGACTCACGGCGCCGGAGTG1140              GCATGAAGCTGTAGGGACTCATCTTGCATGGCACCGCCGGCCAGACACTGCCCAAGGACC1200              AGTATTTGTCACTACCAAACTCAGCCCTTCTTGGAATACAGCCTTTCAAGCAGAGGACAG1260              AAGGGTCCTTCTCCTTATGTGGGAAATGGGCCTAGTAGATGCAGAATTCAAAGATGTCGG1320              TTGGGAGAAACTAGCTCTGATCCTAACAGGCCACGTTAAACTGCCCATCTGGAGAATCGC1380              CTGCAGGTGGGGCCCTTTCCTTCCCGCCAGAGTGGGGCTGAGTGGGCGCTGAGCCAGGCC1440              GGGGGCCTATGGCAGTGATGCTGTGTTGGTTTCCTAGGGATGCTCTAACGAATTACCACA1500              AACCTGGTGGATTGAAACAGCAGAACTTGATTCCCTTACAGTTCTGGAGGCTGGAAATCT1560              GGGATGGAGGTGTTGGCAGGGCTGTGGTCCCTTTGAAGGCTCTGGGGAAGAATCCTTCCT1620              TGGCTCTTTTTAGCTTGTGGCGGCAGTGGGCAGTCCGTGGCATTCCCCAGCTTATTGCTG1680              CATCACTCCAGTCTCTGTCTCTTCTGTTCTCTCCTCTTTTAACAACAGTCATTGGATTTA1740              GGGCCCACCCTAATCCTGTGTGATCTTATCTTGATCCTTATTAATTAAACCTGCAAATAC1800              TCTAGTTCCAAATAAAGTCACATTCTCAGGTAAAAAAAAAAAAAAAAAAAA1851                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 365 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Stomach                                                          (B) CLONE: 214915                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerLeuIleArgLysLysGlyPheTyrLysGlnAspValAsnLys                              151015                                                                        ThrAlaTrpGluLeuProLysThrTyrValSerProThrHisValGly                              202530                                                                        SerGlyAlaTyrGlySerValCysSerAlaIleAspLysArgSerGly                              354045                                                                        GluLysValAlaIleLysLysLeuSerArgProPheGlnSerGluIle                              505560                                                                        PheAlaLysArgAlaTyrArgGluLeuLeuLeuLeuLysHisMetGln                              65707580                                                                      HisGluAsnValIleGlyLeuLeuAspValPheThrProAlaSerSer                              859095                                                                        LeuGlyAsnPheTyrAspPheTyrLeuValMetProPheMetGlnThr                              100105110                                                                     AspLeuGlnLysIleMetGlyMetGluPheSerGluGluLysIleGln                              115120125                                                                     TyrLeuValTyrGlnMetLeuLysGlyLeuLysTyrIleHisSerAla                              130135140                                                                     GlyValValHisArgAspLeuLysProGlyAsnLeuAlaValAsnGlu                              145150155160                                                                  AspCysGluLeuLysIleLeuAspLeuGlyLeuAlaArgHisAlaAsp                              165170175                                                                     AlaGluMetThrGlyTyrValValThrArgTrpTyrArgAlaProGlu                              180185190                                                                     ValIleLeuSerTrpMetHisTyrAsnGlnThrValAspIleTrpSer                              195200205                                                                     ValGlyCysIleMetAlaGluMetLeuThrGlyLysThrLeuPheLys                              210215220                                                                     GlyLysAspTyrLeuAspGlnLeuThrGlnIleLeuLysValThrGly                              225230235240                                                                  ValProGlyThrGluPheValGlnLysLeuAsnAspLysAlaAlaLys                              245250255                                                                     SerTyrIleGlnSerLeuProGlnThrProArgLysAspPheThrGln                              260265270                                                                     LeuPheProArgAlaSerProGlnProAlaAspLeuLeuGluLysMet                              275280285                                                                     LeuGluLeuAspValAspLysArgLeuThrAlaAlaGlnAlaLeuThr                              290295300                                                                     HisProPhePheGluProPheArgAspProGluGluGluThrGluAla                              305310315320                                                                  GlnGlnProPheAspAspSerLeuGluHisGluLysLeuThrValAsp                              325330335                                                                     GluTrpLysGlnHisIleTyrLysGluIleValAsnPheSerProIle                              340345350                                                                     AlaArgLysAspSerArgArgArgSerGlyMetLysLeu                                       355360365                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY:GenBank                                                           (B) CLONE: GI 531125                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetSerGlnGluArgProThrPheTyrArgGlnGluLeuAsnLysThr                              151015                                                                        IleTrpGluValProGluArgTyrGlnAsnLeuSerProValGlySer                              202530                                                                        GlyAlaTyrGlySerValCysAlaAlaPheAspThrLysThrGlyHis                              354045                                                                        ArgValAlaValLysLysLeuSerArgProPheGlnSerIleIleHis                              505560                                                                        AlaLysArgThrTyrArgGluLeuArgLeuLeuLysHisMetLysHis                              65707580                                                                      GluAsnValIleGlyLeuLeuAspValPheThrProAlaArgSerLeu                              859095                                                                        GluGluPheAsnAspValTyrLeuValThrHisLeuMetGlyAlaAsp                              100105110                                                                     LeuAsnAsnIleValLysCysGlnLysLeuThrAspAspHisValGln                              115120125                                                                     PheLeuIleTyrGlnIleLeuArgGlyLeuLysTyrIleHisSerAla                              130135140                                                                     AspIleIleHisArgAspLeuLysProSerAsnLeuAlaValAsnGlu                              145150155160                                                                  AspCysGluLeuLysIleLeuAspPheGlyLeuAlaArgHisThrAsp                              165170175                                                                     AspGluMetThrGlyTyrValAlaThrArgTrpTyrArgAlaProGlu                              180185190                                                                     IleMetLeuAsnTrpMetHisTyrAsnGlnThrValAspIleTrpSer                              195200205                                                                     ValGlyCysIleMetAlaGluLeuLeuThrGlyArgThrLeuPhePro                              210215220                                                                     GlyThrAspHisIleAspGlnLeuLysLeuIleLeuArgLeuValGly                              225230235240                                                                  ThrProGlyAlaGluLeuLeuLysLysIleSerSerGluSerAlaArg                              245250255                                                                     AsnTyrIleGlnSerLeuAlaGlnMetProLysMetAsnPheAlaAsn                              260265270                                                                     ValPheIleGlyAlaAsnProLeuAlaValAspLeuLeuGluLysMet                              275280285                                                                     LeuValLeuAspSerAspLysArgIleThrAlaAlaGlnAlaLeuAla                              290295300                                                                     HisAlaTyrPheAlaGlnTyrHisAspProAspAspGluProValAla                              305310315320                                                                  AspProTyrAspGlnSerPheGluSerArgAspLeuLeuIleAspGlu                              325330335                                                                     TrpLysSerLeuThrTyrAspGluValIleSerPheValProProPro                              340345350                                                                     LeuAspGlnGluGluMetGluSer                                                      355360                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Oligomer R                                                       (B) CLONE:                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetSerGlnGluArgProThrPheTyrArgGlnGluLeuAsnLysThr                              151015                                                                        IleTrpGluValProGluArgTyrGlnAsnLeuSerProValGlySer                              202530                                                                        GlyAlaTyrGlySerValCysAlaAlaPheAspThrLysThrGlyLeu                              354045                                                                        ArgValAlaValLysLysLeuSerArgProPheGlnSerIleIleHis                              505560                                                                        AlaLysArgThrTyrArgGluLeuArgLeuLeuLysHisMetLysHis                              65707580                                                                      GluAsnValIleGlyLeuLeuAspValPheThrProAlaArgSerLeu                              859095                                                                        GluGluPheAsnAspValTyrLeuValThrHisLeuMetGlyAlaAsp                              100105110                                                                     LeuAsnAsnIleValLysCysGlnLysLeuThrAspAspHisValGln                              115120125                                                                     PheLeuIleTyrGlnIleLeuArgGlyLeuLysTyrIleHisSerAla                              130135140                                                                     AspIleIleHisArgAspLeuLysProSerAsnLeuAlaValAsnGlu                              145150155160                                                                  AspCysGluLeuLysIleLeuAspPheGlyLeuAlaArgHisThrAsp                              165170175                                                                     AspGluMetThrGlyTyrValAlaThrArgTrpTyrArgAlaProGlu                              180185190                                                                     IleMetLeuAsnTrpMetHisTyrAsnGlnThrValAspIleTrpSer                              195200205                                                                     ValGlyCysIleMetAlaGluLeuLeuThrGlyArgThrLeuPhePro                              210215220                                                                     GlyThrAspHisIleAsnGlnLeuGlnGlnIleMetArgLeuThrGly                              225230235240                                                                  ThrProProAlaTyrLeuIleAsnArgMetProSerHisGluAlaArg                              245250255                                                                     AsnTyrIleGlnSerLeuThrGlnMetProLysMetAsnPheAlaAsn                              260265270                                                                     ValPheIleGlyAlaAsnProLeuAlaValAspLeuLeuGluLysMet                              275280285                                                                     LeuValLeuAspSerAspLysArgIleThrAlaAlaGlnAlaLeuAla                              290295300                                                                     HisAlaTyrPheAlaGlnTyrHisAspProAspAspGluProValAla                              305310315320                                                                  AspProTyrAspGlnSerPheGluSerArgAspLeuLeuIleAspGlu                              325330335                                                                     TrpLysSerLeuThrTyrAspGluValIleSerPheValProProPro                              340345350                                                                     LeuAspGlnGluGluMetGluSer                                                      355360                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Oligomer F                                                       (B) CLONE:                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AAGACATCCAGGAGCCCAATG21                                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: GI 603917                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGGTGATCCTCAGCTGGATGCAC23                                                     __________________________________________________________________________

We claim:
 1. A purified polynucleotide encoding a polypeptide with anamino acid sequence shown in SEQ ID NO:2.
 2. The polynucleotide of claim1 wherein the nucleic acid sequence comprises SEQ ID NO:1, or itscomplement.
 3. A method for detecting the polynucleotide encoding thehuman MAP kinase homolog of claim 1 in a biological sample comprisingthe steps of:(a) hybridizing a polynucleotide which is complementary tothe sequence of SEQ ID: 1 to nucleic acid material of a biologicalsample, thereby forming a specific hybridization complex, and (b)detecting said hybridization complex, wherein the presence of saidcomplex correlates with the presence of a polynucleotide encoding saidMAP kinase homolog in said biological sample.
 4. An expression vectorcomprising the polynucleotide of claim
 1. 5. A host cell transformedwith the expression vector of claim 4.